Functionality and Protein Structure - American Chemical Society

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Conformation and Functionality of M i l k Proteins C. V. MORR Department of Food Science, Clemson University, Clemson, SC 29631

We are indebted to numerous researchers who have contributed sufficient basic knowledge to enable us to unravel the complexities of the milk protein system, e.g., the caseins and whey proteins and their subfractions. These findings have been collected, evaluated and organized into a comprehensive report by the "Milk Protein Nomenclature Committee" (1). This latter report contains excellent details on the primary structure and physicochemical properties of most of the casein monomer subunits and the whey proteins, as well as for most of their genetic polymorphs. Milk proteins are widely recognized for their superior nutritional, organoleptic, and functional properties, as components of milk and in milk-containing food formulations. Although the caseinates have superior functional properties, especially for stabilizing emulsions and foams, there is a need to improve the functionality of the whey protein concentrates. For example, the whey protein concentrates generally exhibit poor functional properties in food applications requiring high solubility. This paper draws heavily upon the "Nomenclature Committee Report" (1) as well as several recent comprehensive reports that have considered the primary structure and conformation of the casein monomer subunits and how they are assembled into submicellar aggregates and casein micelles (2, 3) . These basic relationships were utilized to develop additional projections relating to the conformation and functional properties of the major milk proteins, e.g., commercial caseinates and whey protein concentrates in food applications. Fractionation and Distribution of Major Milk Proteins. The fractionation scheme and distribution data for the major milk proteins and their subfractions are given in Figure 1 (1). Although the caseins are easily separated from whey proteins by adjusting milk to pH 4.6-5.0, further separation and purification of the individual caseins is extremely difficult, due to their strong interaction, and requires the most sophisticated protein 0-8412-0478-0/79/47-092-065$05.00/0 © 1979 American Chemical Society

66

F U N C T I O N A L I T Y A N D PROTEIN STRUCTURE

f r a c t i o n a t i o n techniques a v a i l a b l e . S i m i l a r l y , the i n d i v i d u a l whey p r o t e i n components a r e s e p a r a t e d a n d p u r i f i e d o n l y b y a p p l i cation of elaborate fractionation techniques.


Nomenclature

and P h y s i c o - c h e m i c a l P r o p e r t i e s o f the C a s e i n s .

The f o u r m a j o r c a s e i n f r a c t i o n s c o n t a i n s u b f r a c t i o n s a n d g e n e t i c polymorphs (Table 1 ) , each of which p o s s e s s e s a unique p r i mary s t r u c t u r e a n d a s s o c i a t e d p h y s i c o - c h e m i c a l p r o p e r t i e s (1). F o r e x a m p l e , α - c a s e i n c o n t a i n s one m a j o r s u b f r a c t i o n (α - - C n ) p l u s s e v e r a l minor components. The f o u r g e n e t i c v a r i a n t s o f a i ~ c a s e i n have s i m i l a r i s o i o n i c p o i n t s , which would be e x p e c t e d from t h e i r s i m i l a r primary s t r u c t u r e s . The β - c a s e i n s , κ - c a s e i n s , a n d "whole α - c a s e i n s " have a l s o been f r a c t i o n a t e d and t h e i r s u b f r a c tions c h a r a c t e r i z e d (Table 1 ) . The i s o i o n i c p o i n t s f o r a l l o f these c a s e i n s u b u n i t s range from about 4.9 to 6.0 and t h e i r mole c u l a r w e i g h t s range from about 11,500 t o 2 4 , 0 0 0 . s

P r i m a r y S t r u c t u r e o f t h e M a j o r C a s e i n Monomer

s

Subunits.

The p r i m a r y s t r u c t u r e s ( F i g u r e s 2, 3 and 4) a r e g i v e n f o r a - g ^ e a s e i n , β - c a s e i n , a n d κ - c a s e i n (I). The most s i g n i f i c a n t a s pects o f these primary s t r u c t u r e s are t h e i r d i s p r o p o r t i o n a t e d i s tribution o f a c i d i c amino a c i d s , serine phosphate g r o u p s a n d h y d r o p h o b i c amino a c i d s a l o n g t h e p o l y p e p t i d e c h a i n . As d e m o n s t r a t e d b y B l o o m f i e l d a n d Mead ( 2 ) , t h e u n e v e n d i s t r i b u t i o n o f amino a c i d s f o r t h e m a j o r c a s e i n monomer s u b u n i t s l e a d s t o h i g h l y charged ( n e g a t i v e ) r e g i o n s , which a r e s e p a r a t e d from the s t r o n g l y hydrophobic r e g i o n s , a l o n g the p o l y p e p t i d e c h a i n s . These amphophilic molecules are h i g h l y susceptible to intermolecular i n t e r a c t i o n and p o l y m e r i z a t i o n through the formation o f hydrophobic a n d i o n i c , e s p e c i a l l y Ca b o n d s . S l a t t e r y (3) u s e d t h i s a n d o t h e r r e l e v a n t i n f o r m a t i o n t o d e v e l o p c o n f o r m a t i o n a l models f o r each o f t h e m a j o r c a s e i n monomer s u b u n i t s . I n h e r e n t i n t h e s e models i s the c o n s i d e r a t i o n t h a t the u n i f o r m d i s t r i b u t i o n o f p r o l i n e a l o n g t h e p o l y p e p t i d e c h a i n r e s u l t s i n a random c o i l , w i t h l i t t l e h e l i cal structure. The m o d e l f o r a - c a s e i n t h a t b e s t f i t s t h e d a t a i s a c o m p a c t , p r o l a t e e l l i p s o i d c o n t a i n i n g most o f t h e h y d r o p h o b i c amino a c i d r e s i d u e s , p l u s a 40 amino a c i d r e s i d u e c h a i n c o n t a i n i n g most o f t h e a c i d i c amino a c i d s a n d s e r i n e p h o s p h a t e s that e x t e n d i n t o t h e aqueous p h a s e as a l o o p ( 3 ) . The compact h y d r o p h o b i c r e g i o n i s s t a b i l i z e d by i n t r a m o l e c u l a r hydrophobic b o n d i n g , whereas the e x p o s e d , a c i d i c loop i s r e a d i l y a c c e s s i b l e f o r i n t e r a c t i o n w i t h adjacent c a s e i n molecules through formation o f i o n i c , mainly Ca, bonds. The m o d e l d e v e l o p e d b y S l a t t e r y (3) f o r β - c a s e i n a l s o c o n t a i n s a compact h y d r o p h o b i c r e g i o n , w i t h a b o u t t h e same d i m e n s i o n s a s f o r a - c a s e i n , a n d w i t h a s t r o n g l y a c i d i c p o l y p e p t i d e c h a i n , e . g . , amino a c i d r e s i d u e s 1 - 2 5 , e x p o s e d t o t h e aqueous m e d i a . The m o d e l f o r κ - c a s e i n ( 3 ) a l s o c o n t a i n s a compact h y d r o p h o b i c r e g i o n , as a b o v e , p l u s a f r e e l y e x p o s e d p o l y p e p t i d e c h a i n c o n t a i n i n g a h i g h p o r t i o n o f a c i d i c amino a c i d s as w e l l a s s

s

23-35 3-7

(S-Caseins

γ-Caseins

Adapted from Whitney et a l . , 1976

""Calculated from primary structure where

possible

^ T o t a l of genetic v a r i a n t s and minor components

a

4

2-6

Proteose peptones

4

1.9-3.3

Immunoglobulins

2

1

2-5

.7-1.3

4

9

3.30-3.7

5.50-8.3

4.20-4.5

5.13

5.35-5.41

5.80-6.0

5.20-5.85

4,100-40,800

150,000-1 m i l l i o n

14,146-14,174

66,500-69,000

18,275-18,362

11,556-20,629

23,939-24,089

19,005-19,037

5.37

2 7

22,068-22,723

0

Molecular weight

4.92-5.35

Isoionic point

3

9

Number of components**

0. -Lactalbumins

Bovine serum albumin

3-Lactogiobulins

Whey Proteins 7-12

8-15

K-Caseins

s

45-55

Approx % of skimmilk protein

Amount and Selected P r o p e r t i e s of M i l k Proteins and Their Components

a -Caseins

Caseins

Protein

Table I .


FUNCTIONALITY

A N D PROTEIN

STRUCTURE

SKIMMILK pH 4.6 @ 20 C ι

1

PRECIPITATE

SUPERNATANT

I

I


CASEINS (75-85 %)

WHEY PROTEINS (15-22 %)

a -

(45-55)

BSA

(.7-1.3)

β-

(25-35)

£-Lg

(7-12)

K-

(8-15)

a-La

(2-5)

y-

(3-7)

Ig (1.9-3.3)

g

PP (2-6) Figure 1.

Fractionation and distribution of the major milk proteins

10 20 H.Arg-Pro- L y s - H i s - P r o - I le - Lys-His- G l n - G l y - L e u - P r o - G l n - | G l u - V a l - L e u - A s n - G l u - A s n - L e u Absent in variant A 30 40 Leu-Arg-Phe-Phe-Val-Ala|-Pro-Phe-Pro-Gln-Val- Phe-Gly-Lys-Glu-Lys-Val -Asn-Glu-Leu50 60 Ser—Lys-Asp-I le - G l y - S e r - G l u - S e r - T h r - G l u - A s p - G i n 4-Ala Met-Glu-Asp-lle - Lys-Glu-MetP Ρ ThrP (variant D) 70 80 G l u - A l a - G l u - S e r - l l e - S e r - S e r - S e r - G l u - G l u - l l e - Val-Pro-Asn-Ser —Val—Glu - G i n - L y s - H i s Ρ Ρ Ρ Ρ Ρ 90 100 Ile - G l n - L y s - G l u - A s p - V a l - P r o - S e r - G l u - A r g - T y r - L e u - G l y - T y r - L e u - G l u - G l n - L e u - L e u - A r g L

110 120 Leu -Lys -Lys -Ty r-Lys -Val - Pro - G i n - L e u -Glu-Ile - Val -Pro -Asn -Ser - Ala - G l u - G l u - A r g - L e u P 130 140 His-Ser-Met-Lys-GIn-Gly-lle - His-Ala-GIn-GIn-Lys-Glu-Pro-Met-Ile - Gly-Val-Asn-GIn150 160 Glu-Leu^Ala-Tyr-Phe-Tyr-Pro-Glu-Leu-Phe-Arg-GIn-Phe-Tyr-GIn-Leu-Asp-Ala-Tyr-Pro170 180 Ser-Gly-Ala-Trp-Tyr-Tyr-Val-Pro-Leu-Gly-Thr-GIn-Tyr-Thr-Asp-Ala-Pro-Ser-Phe-Ser190 199 A s p - l l e - Pro- Asn-Pro-Me - Gly-Ser -Glu-Asn-Ser-|Glu|- Lys -Thr - T h r - M e t - P r o - Leu-Trp.OH Gly (variant Cî

Journal of Dairy Science Figure 2.

Primary structure of bovine a -Cn-B s

(1)

MORR

Milk

Proteins

10 20 H.Arg-Glu-Leu-Glu-Glu-Leu^sn-Val-Pro-Gly-Glu-lle - Val-Glu-Ser-Leu-Ser-Ser -Ser-Glu Ρ Ρ Ρ Ρ 7, -caseins 30 40 ^,μ-^, - .... - ™ - . , - u . - He - G l u - L y s - Phe - GI n- Ser-fcluV|Glul-Gln-Gln-GIn(absent in variant C) Ρ Lys Lys (variante) (variant E) 50 60 Thr-Giu-Asp-Glu-Leu-GIn-Asp-Lys-lIe - His-Pro-Phe-Ala-Gln-Thr-GIn-Ser -Leu-Val - T y r -


y

c

L y 3

C y a

70 80 P r o - P h e - P r o - G l y - P r o - I l e -| Prof- A s n - S e r - L e u - P r o - G I n - A s n - l l e - P r o - P r o - L e u - T h r - G i n - T h r (variants C, A , and B) His 90 100 Pro-Val - V a l - V a l - P r o - P r o - P h e - L e u - G I n - P r o - G l u - V a l - M e t - G l y - V a l - S e r - L y s - V a l - L y s - G l u 1

! •γ3-caseins (R-, TS-B) 110 120 A la - Met - A la - Pro - LvsrfHTi - Lvs - GI u - Met -Pro - Phe - P r o - Lys - T y r - P r o - V a l - G l n - P r o - P h e - T h r Gln (variant A ) I -caseins (S-, T S - A ) 130 140 Glu-jSerf-GIn-Ser - Leu - T h r - Leu -Thr - A s p - V a l - Glu - Asn - L e u - H i s - L e u - P r o - P r o - L e u - L e u - L e u Arg (variant B) 150 160 Gin- Ser - T r p -Met- His - G i n - Pro - H i s - G l n - P r o - Leu-Pro - P r o - T h r - V a l - Met- Phe-Pro - P r o - G i n 3

2

170 180 Ser-Val—Leu-Ser- Leu-Ser-GIn-Ser-Lys-Val-Leu-Pro - V a l - P r o - G l u - L y s - A l a - V a l - P r o - T y r -

P r o - G I n - A r g - A s p - M e t - P r o -Ile -

Val-Arg-Gly-Pro-Phe-Pro-Ile

190 200 Gln-Ala-Phe-Leu-Leu-Tyr-GIn-GIn-Pro-Val-Leu-Gly-Pro-

- Ile

209 -Val.OH


Primary structure of bovine β-Cn-A

2

PyroGlu-Glu-GIn-Asn-GIn-Glu-GIn-Pro (Gln) (Glu) (Glu)

(1)

10 20 -Ile - A r g - C y s - G l u - L y s - A s p - G l u - A r g - P h e - P h e - S e r - A s p -

30 40 Lys -Ile - A l a - L y s - T y r - I l e - P r o - H e - G l n - T y r - V a l - L e u - S e r - A r g - T y r - P r o - S e r - T y r - G l y - L e u 50 60 A s n - T y r - T y r - G l n - G l n - L y s -Pro - V a l - A l a - L e u - l l e - A s n - A s n - G I n - P h e - L e u - P r o - T y r - P r o - T y r 70 80 Tyr-Ala - L y s - P r o - A l a - A l a - V a l - A r g - S e r - P r o - A l a - G I n - l l e - Leu-GIn-Trp-GIn-Val- Leu-Ser90 100 Asp -Thr - V a l - P r o - A l a - L y s - S e r - C y s - G l n - A l a - G I n - P r o - T h r - T h r - M e t - A l a - A r g - H i s - P r o - H i s (Asn) (Pro) 1051106 110 120 Pro - H i s - L e u - S e r - P h e - M e t - A l a - I le - P r o - P r o - L y s - L y s - A s n - G I n - A s p - L y s - T h r - G l u - l l e - P r o mis) 130 136 140 Thr-lle - Asn-Thr-lle - A l a - S e r - G l y - G t u - P r o -Thr-Ser -Thr-Pro-Thr-ffuj- G l u - A l a - V a l - G l u I Thr (variant A) NeuNAc(2-»3(6)) Gal(l->-3(6))GalNAc (carbohydrate-containing κ-caseins) 148 1 50 1 60 Ser - T h r - V a l - A l a - T h r - L e u - G l u - p U â r - S e r - P r o - G l u - V a l - l l e - G l u - S e r - P r o - P r o - G l u - I l e - A s n (variant A) Asp Ρ 169 Thr-Val-Gin-Val-Thr-Ser-Thr-Ala-Val.OH


Primary structure of bovine κ-Cn-B (l)

70

F U N C T I O N A L I T Y A N D PROTEIN STRUCTURE

t h e n e g a t i v e l y c h a r g e d g l y c o m a e r o p e p t i d e (GMP) g r o u p . It is be l i e v e d t h a t the exposed, GMP-containing p o l y p e p t i d e c h a i n i s r e s p o n s i b l e f o r κ - c a s e i n s pronounced s t a b i l i z i n g a c t i o n f o r the m i l k c a s e i n m i c e l l e s y s t e m , s i n c e c l e a v a g e o f t h i s e n t i t y by e n z y m a t i c r e n n e t m o d i f i c a t i o n c o m p l e t e l y removes i t s s t a b i l i z i n g ability. C o m p a r i s o n o f t h e d i s t r i b u t i o n o f t h e number o f i m p o r t a n t ami n o a c i d t y p e s f o r e a c h o f t h e c a s e i n s ( T a b l e 2) p r o v i d e s an i n d i c a t i o n o f t h e i r o v e r a l l c a p a c i t y f o r a g g r e g a t i o n a n d i n t e r a c t i o n v i a h y d r o p h o b i c , i o n i c and d i s u l f i d e bond f o r m a t i o n . I t w i l l be n o t e d t h a t κ - c a s e i n i s t h e o n l y monomer s u b u n i t t h a t c o n t a i n s a d i s u l f i d e group, and t h i s i s undoubtedly r e s p o n s i b l e f o r i t s a b i l i t y t o s e l f a g g r e g a t e as w e l l as t o i n t e r a c t w i t h β l a c t o g l o b u l i n during heat p r o c e s s i n g of m i l k .


1

Association of

C a s e i n Monomer

Subunits.

A l l t h r e e o f t h e c a s e i n monomer s u b u n i t s e x h i b i t a s t r o n g c a p a c i t y to i n t e r a c t at n e u t r a l pH. A d d i t i o n o f Ca i o n s s t r o n g l y promotes t h e i r tendency to a g g r e g a t e , presumably by c r o s s - l i n k i n g c a r b o x y l and phosphate e s t e r groups l o c a t e d i n t h e i r exposed, a c i d i c peptide c h a i n s . A d d i t i o n o f CA p r o b a b l y a l s o p r o m o t e s a g g r e g a t i o n o f t h e c a s e i n monomer s u b u n i t s by r e d u c i n g t h e i r molecular charge. I t i s p o s s i b l e t h a t , due t o s t e r i c h i n d r a n c e , κ - c a s e i n ' s n e g a t i v e l y c h a r g e d GMP group may be u n a b l e t o p a r t i c i p a t e i n i n t e r m o l e c u l a r Ca c r o s s - l i n k s w i t h a d j a c e n t c a s e i n monomers a n d a g g r e g a t e s . T h i s may a c c o u n t f o r t h e m i c e l l e s t a b i l i z i n g f u n c t i o n d i s c u s s e d above f o r κ - c a s e i n . These c a s e i n - c a s e i n i n t e r a c t i o n s , e s p e c i a l l y those i n v o l v i n g β - c a s e i n , are s t r o n g l y t e m p e r a t u r e - d e p e n d e n t ; a phenomenon t h a t i s i m p o r t a n t f o r a s s o c i a t i o n / d i s s o c i a t i o n o f t h e s e monomer s u b u n i t s w i t h and f r o m t h e milk c a s e i n m i c e l l e system (4). κ - c a s e i n a l s o c o n t a i n s two h Cys r e s i d u e s p e r monomer s u b u n i t a n d i s t h u s c a p a b l e o f i n t e r a c t i n g w i t h t h e whey p r o t e i n s , e.g., m a i n l y β - l a c t o g l o b u l i n , v i a the d i s u l f i d e i n t e r c h a n g e mechanism a t t e m p e r a t u r e s a t o r above 6 5 ° C . T h i s l a t t e r phenomenon i s b e l i e v e d t o be i m p o r t a n t i n p r o v i d i n g c o l l o i d a l s t a b i l i t y t o the m i l k c a s e i n m i c e l l e s y s t e m , as w e l l a s t o t h e whey p r o t e i n s , i n h i g h temperature processed m i l k p r o d u c t s . I t has a l s o been p o s t u l a t e d t h a t t h i s l a t t e r i n t e r a c t i o n w i t h 3 - l a c t o g l o b u l i n may a l t e r t h e a v a i l a b i l i t y o f κ - c a s e i n i n the m i c e l l e , a n d t h u s h a s a d e t r i m e n t a l e f f e c t upon t h e c h e e s e m a k i n g p r o p e r t i e s o f m i l k (4). The C a s e i n M i c e l l e

System.

The m a j o r c a s e i n s e x i s t i n m i l k as h i g h l y s t r u c t u r e d , s p h e r i c a l a g g r e g a t e s , c o n s i s t i n g o f 450 t o 1 0 , 0 0 0 s u b u n i t s (3), commonly r e f e r r e d t o as m i c e l l e s . The i m p o r t a n t p h y s i c o - c h e m i c a l p r o p e r t i e s o f t h e m i c e l l e s a r e s u m m a r i z e d i n T a b l e 3. Casein m i c e l l e s a r e s y n t h e s i z e d i n v i v o by b i o c h e m i c a l l y c o n t r o l l e d p r o c e s s e s , w h i c h have not been t o t a l l y c h a r a c t e r i z e d (5). Even

si

A r g and H i s

1 2

20

17

1

25

169

Lys,

0

0

35

20

5

25

209

0

STÂLIC ACID

0

h CYS

PROLINE

17

2

25

BASIC

8

SERINE Ρ

Monomers

42

1

Casein

the Major

199

G l u and T y r

2

-Β

ACIDIC

Amino A c i d C o m p o s i t i o n o f

TOTAL

II.

Asp,

K-B

3-A

α

CASEIN

Table


III.

nm

2

Q

w

Adapted

from M o r r ,

25 C

1975

cc/g micelle 3.5-6

2-3

Q

S

1.6-2

dry micelle

ο

2-18 χ 10

8-22 χ 10

C

Voluminosity,

a

S

Daltons

g water/g

Weight,

Constant,

100-250

25 C

0-5

Solvation,

Molecular

Sedimentation

Diameter,

95-98 %

in Milk

25 C

of C a s e i n M i c e l l e s

75-80 %

micelle/total

Physico-Chemical Properties

C

0-5

Casein,

Table a


S

°

>

3

g

π


4.

MORR

Milk

73

Proteins

though they a r e remarkably s t a b l e i n m i l k under normal c o n d i t i o n s , t r e a t m e n t s s u c h as c o o l i n g a n d w a r m i n g ( b e t w e e n 5 a n d 37 C ) , a d j u s t i n g t h e pH b y c h e m i c a l o r m i c r o - b i o l o g i c a l m e a n s , adjusting Ca a n d o t h e r i o n c o n c e n t r a t i o n s , h e a t i n g , e v a p o r a t i o n a n d d r y i n g , e n z y m a t i c m o d i f i c a t i o n (as w i t h r e n n e t ) a l l e f f e c t t h e s t a b i l i t y o f the m i c e l l e s ( 4 ) . T h e r e h a v e b e e n a number o f excellent p a p e r s p u b l i s h e d t h a t p r o v i d e an i n s i g h t i n t o t h e c o m p l e x i t y o f t h e m i l k c a s e i n m i c e l l e s t r u c t u r e as w e l l as a n u n d e r s t a n d i n g o f the f a c t o r s t h a t s t a b i l i z e i t . S c i e n t i s t s who h a v e made s p e c i a l c o n t r i b u t i o n s i n t h i s r e g a r d a r e : Waugh, e t a l . ( 6 ) , P a y e n s (7), G a m i e r a n d R i b a d e a u - D u m a s ( 8 ) , M o r r ( 9 ) , Rose (10) a n d B l o o m f i e l d and Mead ( 2 ) . S l a t t e r y (3) r e c e n t l y p r o p o s e d a m o d e l f o r t h e m i c e l l e s a n d t h e i r m e c h a n i s m o f f o r m a t i o n , b a s e d upon t h e a m p h o p h i l i c n a t u r e o f t h e i n d i v i d u a l c a s e i n monomer s u b u n i t s a n d their submicellar aggregates. A l t h o u g h S l a t t e r y s model c o n s i ders the r o l e o f the s u b m i c e l l a r a g g r e g a t e s o f the t h r e e major caseins ( a - , β - , and κ - c a s e i n ) and t h e i r assembly mechanism, it i g n o r e s the importance and i r r e v e r s i b l e s t r u c t u r e o f " c o l l o i d a l p h o s p h a t e " (11) i n t h e m i c e l l e . 1

g

Commercial

Caseinates.

C a s e i n i s i s o l a t e d f r o m m i l k a n d p r o d u c e d as N a , K , a n d Ca c a s e i n a t e s by the g e n e r a l s t e p s o u t l i n e d i n F i g u r e 5. Both the a c i d i f i c a t i o n and n e u t r a l i z a t i o n s t e p s p r o f o u n d l y a f f e c t the s t r u c t u r e and assembly o f r e s u l t i n g c a s e i n a t e p a r t i c l e s . These d e r i v e d c a s e i n a t e aggregate p a r t i c l e s have l i t t l e s i m i l a r i t y to milk casein m i c e l l e s . A c i d i f i c a t i o n to i s o e l e c t r i c c o n d i t i o n s , e . g . , pH 4 . 5 - 5 . 0 , c o m p l e t e l y d i s s i p a t e s t h e c o l l o i d a l p h o s p h a t e s t r u c t u r e a n d f r e e s t h e c a s e i n p r e c i p i t a t e o f Ca a n d o t h e r i n o r ganic i o n s . N e u t r a l i z a t i o n o f the c a s e i n p r e c i p i t a t e r e s o l u b i l i zes the c a s e i n , p r e s u m a b l y by a l t e r i n g i t s charge s u f f i c i e n t l y t o overcome i n t e r m o l e c u l a r h y d r o p h o b i c b o n d i n g . The r e s u l t i n g c a s e i n a t e s y s t e m c o n t a i n s a g g r e g a t e s o f t h e monomer s u b u n i t s i n a range o f s i z e s , depending upon temperature and e l e c t r o l y t e composition (12). The o r i e n t a t i o n o f t h e i n d i v i d u a l c a s e i n mono mer s u b u n i t s w i t h i n t h e s e a g g r e g a t e s i s unknown, b u t i t may b e assumed t h a t , due t o t h e r a p i d i t y o f t h e a c i d i f i c a t i o n / r e s o l u b i l i z a t i o n r e a c t i o n s , they are randomly a s s o c i a t e d . B a s e d upon t h e s t r o n g s e n s i t i v i t y o f t h e s e a g g r e g a t e s t o pH a n d i o n i c a d j u s t ments, as w e l l as t o e n z y m a t i c m o d i f i c a t i o n , i t i s l i k e l y t h a t t h e s u b u n i t s a r e a r r a n g e d w i t h an o r i e n t a t i o n t h a t p r o v i d e s t h e i r h y d r o p h i l i c , a c i d i c - p o l y p e p t i d e - c h a i n s e q u e n c e maximum c o n t a c t w i t h t h e a q u e o u s medium. C o m m e r c i a l c a s e i n a t e s , p r e p a r e d as N a a n d Κ c a s e i n a t e s e x h i b i t i m p r o v e d s o l u b i l i t y and f u n c t i o n a l i t y compared t o Ca c a s e i n a t e . T h i s l a t t e r phenomenon i s p r o b a b l y due t o l a r g e r s i z e d a n d more s t r o n g l y i n t e r a c t i n g Ca c a s e i n a t e a g g r e g a t e s due t o c r o s s - l i n k i n g b y t h e d i v a l e n t c a t i o n s . Comparison o f the above c o m m e r c i a l c a s e i n a t e s w i t h m i l k c a s e i n m i c e l l e s i n d i c a t e s t h a t , under normal food p r o c e s s i n g c o n d i -


74

FUNCTIONALITY AND PROTEIN STRUCTURE

t i o n s , e . g . , pH 6 t o 7, c a s e i n a t e a g g r e g a t e s a r e s m a l l e r a n d more s e n s i t i v e t o pH a n d i o n i c f l u c t u a t i o n t h a n a r e t h e c o l l o i d a l phosphate-containing milk micelles. F o r example, the m i l k m i c e l l e s y s t e m s t a b i l i z e s a h i g h e r l e v e l o f Ca i o n s t h a n c a n t h e commer c i a l c a s e i n a t e s , due t o i t s i n c o r p o r a t i o n i n t o t h e r e l a t i v e l y n o n r e a c t i v e c o l l o i d a l phosphate polymer s t r u c t u r e (10). Although t h e r e h a s b e e n much c o n j e c t u r e on t h e s u b j e c t , i t i s c o n s i d e r e d h i g h l y d o u b t f u l that m i c e l l e s resembling those i n milk i n every d e t a i l , can be r e f o r m e d by c o m b i n i n g t h e i r i n d i v i d u a l components. I f t h i s l a t t e r f u n c t i o n c o u l d be a c c o m p l i s h e d , i t w o u l d p r o b a b l y p e r m i t t h e d e v e l o p m e n t o f new c a s e i n a t e forms w i t h m o d i f i e d f u n c tional properties. Functional Properties

of

Commercial

Caseinates.

Although commercial c a s e i n a t e s are used i n a wide v a r i e t y o f a p p l i c a t i o n s w i t h i n the f o o d i n d u s t r y (13, 14), they are e s p e c i a l l y u s e f u l i n those that u t i l i z e t h e i r e x c e l l e n t s u r f a c t a n t p r o perties. The p r o b a b l e e x p l a n a t i o n f o r t h e e x c e l l e n t surfactant p r o p e r t i e s o f the c a s e i n a t e s l i e s i n t h e i r unique a m p h i p h i l i c c o n f o r m a t i o n as w e l l a s t h e i r o r d e r e d a g g r e g a t e s t r u c t u r e s , as d e s c r i b e d above. I t has been r e p o r t e d t h a t c a s e i n m i c e l l e s r a p i d l y a s s o c i a t e on t h e s u r f a c e o f f r e s h l y - f o r m e d f a t g l o b u l e s (15) to s t a b i l i z e them a g a i n s t c o a l e s c e n c e a n d s e p a r a t i o n . T h u s , i t may be p o s t u l a t e d t h a t c a s e i n a t e , e i t h e r as monomer ^ u n i t s o r t h e i r a g g r e g a t e s , a l s o r a p i d l y m i g r a t e t o a n d a s s o c i a t e on f r e s h l y f o r m e d a i r / w a t e r o r o i l / w a t e r i n t e r f a c e s o f foams o r e m u l s i o n s t o s t a b i l i z e them a g a i n s t c o l l a p s e o r c o a l e s c e n c e . It i s further p r o p o s e d t h a t s u c h e m u l s i o n s a n d f o a m s , due t o t h e h i g h a v a i l a b i l i t y o f c a s e i n a t e h y d r o p h i l i c , a c i d i c p e p t i d e c h a i n s to the a q u e o u s p h a s e , w o u l d e x h i b i t a s t r o n g s u s c e p t i b i l i t y t o pH a n d Ca i o n fluctuations. A l s o , a d d i t i o n a l e m u l s i f i e r s , foaming agents and f o o d i n g r e d i e n t s t h a t a l t e r t h e a v a i l a b i l i t y and r e a c t i v i t y o f t h e exposed c a s e i n a c i d i c c h a i n s would l i k e l y have a p r o f o u n d i n f l u e n c e upon t h e f u n c t i o n a l p r o p e r t i e s o f t h e c a s e i n a t e s i n foam a n d e m u l sion applications. s u

Enzymatic M o d i f i c a t i o n of

Casein.

The a c t i o n o f r e n n e t u p o n c a s e i n m i c e l l e s i n m i l k i l l u s t r a t e s t h e t r e m e n d o u s i m p o r t a n c e o f e n z y m a t i c m o d i f i c a t i o n upon t h e c o n f o r m a t i o n , p h y s i c o - c h e m i c a l and f u n c t i o n a l p r o p e r t i e s o f c a s e i n ates. R e n n e t s p e c i f i c a l l y h y d r o l y z e s t h e 105-106 l i n k a g e o f t h e κ - c a s e i n p o l y p e p t i d e c h a i n w h i c h , although l o c a t e d w i t h i n the m i c e l l e , i s h i g h l y a c c e s s i b l e t o the enzyme. The e f f e c t o f t h i s r e a c t i o n i s t o r e l e a s e t h e s t r o n g l y a c i d i c GMP f r o m κ - c a s e i n monomer s u b u n i t s , t h e r e b y r e d u c i n g t h e m a g n i t u d e o f t h e n e g a t i v e c h a r g e o n t h e m i c e l l e s u f f i c i e n t l y t o p e r m i t them t o associate into a gel structure (4).

4.

MORR

Milk

Proteins

75


P r o p e r t i e s o f t h e Whey P r o t e i n s

and T h e i r

Subfractlons.

As w i t h t h e c a s e i n s , whey p r o t e i n s h a v e b e e n f u r t h e r f r a c t i o n a t e d a n d t h e s e s u b f r a c t i o n s h a v e b e e n c h a r a c t e r i z e d (I) . The p r i m a r y s t r u c t u r e s o f two o f t h e s e p r o t e i n s , 3 - l a c t o g l o b u l i n a n d α - l a c t a l b u m i n a r e g i v e n i n F i g u r e s 6 a n d 7. Comparison o f these primary s t r u c t u r e s w i t h those o f the caseins i n d i c a t e s s e v e r a l ma j o r differences which account f o r t h e i r d i s t i n c t p h y s i c o - c h e m i c a l and f u n c t i o n a l p r o p e r t i e s . I n c o n t r a s t t o t h e c a s e i n s , whey p r o t e i n s e x h i b i t a r a t h e r u n i f o r m d i s t r i b u t i o n o f a c i d i c / b a s i c and h y d r o p h o b i c / h y d r o p h i l i c amino a c i d s a l o n g t h e i r p o l y p e p t i d e chains. T h u s , t h e y l a c k t h e a m p h i p h i l i c n a t u r e o f t h e c a s e i n mo nomer s u b u n i t s , b u t a r e r a t h e r p r e s e n t i n a c o m p a c t , g l o b u l a r conformation (16), The s u b s t a n t i a l l y l o w e r p r o l i n e c o n t e n t i n t h e whey p r o t e i n m o l e c u l e s a l s o p e r m i t s a g l o b u l a r c o n f o r m a t i o n w i t h a s u b s t a n t i a l h e l i c a l c o n t e n t , which e x p l a i n s t h e i r s t r o n g suscep t i b i l i t y to d e n a t u r a t i o n by h e a t and s i m i l a r treatments ( 4 ) . Iso i o n i c p o i n t s o f t h e m a j o r whey p r o t e i n s r a n g e f r o m 4 . 2 t o 5 . 4 (Table 1 ) , which a r e comparable to those o f the c a s e i n s . D e n a t u r a t i o n o f whey p r o t e i n s h a s b e e n shown t o u n f o l d t h e i r p o l y p e p t i d e chains and c o n v e r t t h e i r conformation from a g l o b u l a r to an extended form t h a t f a c i l i t a t e s t h e i r s e l f i n t e r a c t i o n ( a g g r e g a t i o n ) and i n t e r a c t i o n w i t h κ - c a s e i n by d i s u l f i d e i n t e r c h a n g e , i o n i c and hydrophobic bonding ( 4 ) . A l t h o u g h β - l a c t o g l o b u l i n e x i s t s as a monomer w i t h a m o l e c u l a r w e i g h t o f 18,000 a t p H ' s below 3 . 5 , i t a s s o c i a t e s t o form a n octamer w i t h m o l e c u l a r w e i g h t o f 144,000 a t pH*s i n the 3.7 to 5.1 range a n d e x i s t s as a dimer w i t h a m o l e c u l a r w e i g h t o f 36,000 a t pH 5 . 1 (16) . T h e s e p H - d e p e n d e n t i n t e r a c t i o n s a r e due t o c h a n ges i n i o n i z a t i o n o f a c i d i c a n d b a s i c amino a c i d s w h i c h h a v e a n e f f e c t upon t h e f o r m a t i o n a n d d i s s i p a t i o n o f h y d r o p h o b i c a n d d i s u l f i d e bonds. A l t h o u g h 3 - l a c t o g l o b u l i n and α - l a c t a l b u m i n a r e c a p a b l e o f the above a s s o c i a t i o n / d i s s o c i a t i o n r e a c t i o n s , t h e y p r o b a b l y h a v e l i t t l e importance i n determining f u n c t i o n a l p r o p e r t i e s i n food a p p l i c a t i o n s i n t h e pH 6 t o 7 r a n g e . H o w e v e r , p r o t e i n c o n c e n t r a t i o n , pH a n d o t h e r r e l a t e d f a c t o r s do a f f e c t t h e i r s u s c e p t i b i l i t y t o h e a t denaturation (17). P r e p a r a t i o n o f Whey P r o t e i n

Concentrates.

I n c o n t r a s t t o t h e c a s e i n s , whey p r o t e i n s r e t a i n t h e i r s o l u b i l i t y i n t h e pH 4 . 5 - 5 . 0 r a n g e , p r o v i d e d t h e y h a v e n o t b e e n denatured. I t i s therefore r e l a t i v e l y d i f f i c u l t t o recover and p u r i f y undenatured p r o t e i n c o n c e n t r a t e s on a commercial s c a l e . P r o c e s s e s t h a t s e p a r a t e t h e whey p r o t e i n s f r o m t h e l o w m o l e c u l a r w e i g h t , n o n p r o t e i n components o f whey h a v e b e e n u s e d w i t h o n l y moderate success t o date ( 1 8 ) . Such p r o c e s s e s u t i l i z e u l t r a f i l t r a t i o n / r e v e r s e o s m o s i s membrane t e c h n o l o g y , g e l f i l t r a t i o n b y t h e basket c e n t r i f u g e t e c h n i q u e , p o l y v a l e n t i o n p r e c i p i t a t i n g agents

76

FUNCTIONALITY

A N D PROTEIN STRUCTURE

SKIMMILK pH 4.6-5.0 CENTRIFUGE


CASEIN

—1

CURD

WHEY

WASH pH 8.5-9.0 (Na,K,Ca) Figure 5.

Procedure for preparation of commercial caseinate

10 H.Leu-lle - V a l - T h r - G l n - T h r - M e t - L y s - G l y - L e u - A s p - l l e

SPRAY

DRY

(A/Dr 20 - Gin-Lys 1 V a l - A l a - G i y - T h r - T r p - T y r -

30 401 S e r - L e u - A l a - M e t - A l a - A l a - S e r - Asp-lle - Ser- L e u - L e u - A s p - A f a - G l n - S e r - A l a - P r o - L e u - Arg)50 60 V a l - T y r - V a l - G l u - G l u - L e u - L y s - P r o - T h r - P r o - G l u - G l y - A s p - L e u - G l u - l l e - Leu-Leu-JGIn-j-Lys(variant C) His ! 70 80 T r p - G l u - A s n fAsp[-G I u - Cys - A la - G In - Lys - Lys -1 le - I l e - A l a - G l u - L y s - T h r - Lys-Ile - P r o - A l a - | Gly (variant B and C) 90 100 V a l - P h e - L y s - L e u - A s p - A l a -Ile - A s n - G l u - A s n - L y s - V a l - L e u - V a l - L e u - A s p - T h r - A s p - T y r -LysI 110 fSHli 120 L y s - T y r - L e u - L e u - P h e - C y s - M e t - G l u - A s n - S e r - A l a - G l u - P r o - G l u - G l n - S e r - L e u f V a l f Cys'-GIn ] (variant B and C) Ala !S~H> " 130 140 "C'fs - Leu -Val - A r g - T h r - P r o - G l u - Val - A s p - A s p - G l u - Ala - L e u - G l u - L y s - Phe - Asp - L y s - Ala - L e u - ^ 150 Lys- Ala - Leu -Pro -Met -His -1 le - Arg - L e u -Ser - Phe - A s n -Pro - T h r - Leu-Gin - G l u - Glu - G i n 162 His-lle.OH

160 Cys -


Primary structure of bovine β-Lg-A

(1)

20

I 10 H . G I u - G l n - L e u - T h r - L y s - C y s - G l u - V a l - P h e -{Argj-Gtu-Leu - L y s - A s p - L e u - L y s - G l y - T y r - G l y - G l y (variant A) Gin 30 40 V a l - S e r - Leu-Pro - G l u - T r p - V a l - C y s - T h r - T h r - P h e - H i s - T h r - S e r - G l y - T y r - A s p - T h r - G l u - A l a -

I 50 60 lie - V a l - G l u - A s n - A s n - G l n - S e r - T h r - A s p - T y r - G l y - L e u - P h e - G l n - l l e - A s n - A s n - L y s - l l e - T r p I 70 I 80 C y s - L y s - A s n - A s p - G l n - A s p - P r o - H i s - S e r - S e r - Asn-lle - C y s - Asn-Ile - S e r - C y s - A s p - L y s - P h e 90 I 100 L,eu-Asn-Asn-Asp-Leu-Thr-Asn- Asn-lle - M e t - C y s - V a l - L y s - L y s - l i e - Leu-Asp-Lys - V a l - G l y 110 J 120 l i e - A s n - T y r - T r p - L e u - A l a -His - L y s - A l a - L e u - C y s - S e r - Glu - L y s - L e u - A s p - G i n - T r p - Leu - C y s 123 Glu-Lys-Leu.OH


Primary structure of a-La-B (1)


4.

MORR

Milk

Proteins

77

(carbοxymethyl c e l l u l o s e , hexametaphosphate, acrylic acid, etc.), e l e c t r o d i a l y s i s , and i o n - e x c h a n g e r e s i n treatments to fractionate and r e c o v e r t h e whey p r o t e i n c o n c e n t r a t e s w h i c h commonly c o n t a i n o n l y 35 t o 50% p r o t e i n o n a d r y w e i g h t b a s i s . Inherent i n each o f t h e s e p r o c e s s e s i s t h e n e c e s s i t y t o c o n c e n t r a t e t h e d i l u t e whey p r o t e i n s o l u t i o n under c o n d i t i o n s t h a t minimize d e n a t u r a t i o n o f t h e h e a t - s e n s i t i v e whey p r o t e i n s . B o t h o f t h e commonly u s e d c o n c e n t r a t i o n p r o c e s s e s , e . g . , vacuum e v a p o r a t i o n and s p r a y d r y i n g , i n t r o d u c e a s m a l l b u t d e f i n i t e amount o f p r o t e i n d e n a t u r a t i o n i n t o the o v e r a l l p r o c e s s (19). Whey p r o t e i n c o n c e n t r a t e p r e p a r a t i o n p r o c e s s e s b a s e d upon h e a t d e n a t u r a t i o n t o i n s o l u b i l i z e a n d p r e c i p i t a t e them f r o m whey have been d e v e l o p e d (20). S u c h whey p r o t e i n c o n c e n t r a t e p r o d u c t s a r e s u i t a b l e f o r a p p l i c a t i o n s i n p a s t a p r o d u c t s (21) due t o t h e i r i n s o l u b i l i t y and l a c k o f s t i c k i n e s s . Functional Properties

of

Whey P r o t e i n

Concentrates.

A l t h o u g h whey p r o t e i n c o n c e n t r a t e s p o s s e s s e x c e l l e n t n u t r i t i o n a l and o r g a n o l e p t i c p r o p e r t i e s , t h e y o f t e n e x h i b i t o n l y p a r t i a l s o l u b i l i t y a n d do n o t f u n c t i o n as w e l l as t h e c a s e i n a t e s for s t a b i l i z i n g a q u e o u s foams a n d e m u l s i o n s ( 1 9 ) . A number o f compo s i t i o n a l and p r o c e s s i n g f a c t o r s are i n v o l v e d which a l t e r the a b i l i t y o f whey p r o t e i n c o n c e n t r a t e s t o f u n c t i o n i n s u c h f o o d formulations. These i n c l u d e : p H , redox p o t e n t i a l , Ca c o n c e n t r a t i o n , heat d e n a t u r a t i o n , enzymatic m o d i f i c a t i o n , r e s i d u a l p o l y phosphate o r o t h e r p o l y v a l e n t i o n p r e c i p i t a t i n g a g e n t s , residual m i l k l i p i d s / p h o s p h o l i p i d s and c h e m i c a l e m u l s i f i e r s (22). I t i s l i k e l y t h a t t h e i n a b i l i t y o f whey p r o t e i n s t o f u n c t i o n as w e l l a s c a s e i n a t e i n s t a b i l i z i n g foams a n d e m u l s i o n s i s due t o c o n f o r m a t i o n a l a n d s t r u c t i o n a l d i f f e r e n c e s i n t h e two p r o t e i n s . I t i s t h e r e f o r e p o s t u l a t e d t h a t whey p r o t e i n s , w h i c h l a c k an a m p h o p h i l i c c o n f o r m a t i o n , do n o t o r i e n t s u f f i c i e n t l y w e l l a t a i r / w a t e r i n t e r f a c e s t o s t a b i l i z e foam o r e m u l s i o n s y s t e m s as effec t i v e l y as caseinates. D e n a t u r a t i o n o f t h e whey p r o t e i n m o l e c u l e , i f p r o d u c e d a t t h e proper stage of the p r o t e i n concentrate i s o l a t i o n / u t i l i z a t i o n p r o c e s s , can improve the f u n c t i o n a l i t y . The i m p r o v e m e n t i n f u n c t i o n a l i t y i s p r o b a b l y due t o a n u n f o l d i n g o f t h e m o l e c u l e t o e x p o s e h y d r o p h o b i c amino a c i d r e s i d u e s , t h u s m a k i n g t h e p r o t e i n more a m p h o p h i l i c and c a p a b l e o f o r i e n t i n g a t the a i r / w a t e r o r o i l / w a t e r interface. However, i f the p r o t e i n c o n c e n t r a t e i s d e n a t u r e d d u r i n g p r e l i m i n a r y p r e p a r a t i o n s t a g e s , e . g . , p r i o r to o r d u r i n g d r y i n g , t h e i r s o l u b i l i t y and r e l a t e d f u n c t i o n a l i t y a r e a d v e r s e l y affected. Summary. The c o n f o r m a t i o n casein

and p h y s i c o - c h e m i c a l

and whey p r o t e i n c o m p o n e n t s ,

properties

and t h e i r

of

the

subfractions,

major are

78

FUNCTIONALITY AND PROTEIN STRUCTURE


r e l a t e d to the f u n c t i o n a l p r o p e r t i e s of commercial c a s e i n a t e and whey p r o t e i n c o n c e n t r a t e s u s e d i n t h e f o o d p r o c e s s i n g i n d u s t r y . The e f f e c t s o f p H , t e m p e r a t u r e , i o n i c e n v i r o n m e n t , h e a t d e n a t u r a t i o n , enzymatic m o d i f i c a t i o n and p r o c e s s i n g are a l s o c o n s i d e r e d i n this regard. The m a j o r c a s e i n monomer s u b u n i t s h a v e random c o i l c o n f o r m a t i o n that f a c i l i t a t e s strong p r o t e i n - p r o t e i n i n t e r a c t i o n v i a h y d r o p h o b i c and i o n i c b o n d i n g . The u n i q u e a m p h i p h i l i c s t r u c t u r e , which a r i s e s from s e p a r a t e l y c l u s t e r e d h y d r o p h o b i c and n e g a t i v e l y c h a r g e d ( a c i d i c a n d e s t e r p h o s p h a t e ) amino a c i d r e s i d u e s a l o n g t h e p o l y p e p t i d e c h a i n , makes them s u s c e p t i b l e t o pH and Ca i o n c o n centration effects. This amphiphilic nature i s probably responsi b l e f o r the e x c e l l e n t s u r f a c t a n t p r o p e r t i e s of commercial c a s e i n ate i n a v a r i e t y of food a p p l i c a t i o n s . The m a j o r whey p r o t e i n s h a v e c o m p a c t , g l o b u l a r conformations, w i t h a s u b s t a n t i a l amount o f h e l i c a l s t r u c t u r e . This conformation i s p r o b a b l y due t o t h e r a t h e r u n i f o r m d i s t r i b u t i o n o f a c i d i c / b a s i c a n d h y c r o p h i l i c / h y d r o p h o b i c amino a c i d s a l o n g t h e i r p o l y p e p t i d e chains. T h e i r s e n s i t i v i t y to h e a t and o t h e r d e n a t u r i n g a g e n t s i s due t o t h e h i g h c o n t e n t o f s u l f h y d r y l / d i s u l f i d e g r o u p s , w h i c h s t a b i l i z e t h e i r d e r i v e d , random c o i l c o n f o r m a t i o n b y t h e d i s u l f i d e bond i n t e r c h a n g e mechanism. Exposure of s u l f h y d r y l , hydrophobic a n d a c i d i c amino a c i d r e s i d u e s d u r i n g d e n a t u r a t i o n makes t h e whey p r o t e i n s s u s c e p t i b l e to s e l f p o l y m e r i z a t i o n and i n t e r a c t i o n w i t h casein, e . g . , mainly κ-casein. Although heat denaturation gene r a l l y r e d u c e s t h e s o l u b i l i t y a n d f u n c t i o n a l i t y o f whey p r o t e i n s , i t can be u t i l i z e d , i f conducted a t the p r o p e r p o i n t i n the p r o c e s s , to improve t h e i r f u n c t i o n a l i t y .


4. MORR

Milk Proteins

79

Literature Cited 1 Whitney, R. McL., Brunner, J. R., Ebner, Κ. Ε., Farrell, Η. Μ., Jr., Josephson, R. V., Morr, C. V., Swaisgood, H. E . , J. Dairy Sci. (1976) 59, 795. 2 Bloomfield, V. Α., Mead, R. J., Jr., J . Dairy Sci. (1975) 58, 795. 3 Slattery, C. W., J. Dairy Sci. (1976) 59, 1547. 4 Morr, C. V., J. Dairy Sci. (1975)58,977. 5 Farrell, Η. M., Jr., J. Dairy Sci. (1973) 56, 1195. 6 Waugh, D. F., Creamer, L. Κ., Slattery, C. W., Dresdner, G. W., Biochem. (1970) 9, 786. 7 Payens, T. A. J., J. Dairy Sci. (1966) 49, 1317. 8 Garnier, J., Ribadeau-Dumas, B., J . Dairy Res. (1970)37,493. 9 Morr, C. V., J. Dairy Sci. (1967) 50, 144. 10 Rose, D., J. Dairy Sci. (1965) 48, 139. 11 Rose, D., Dairy Sci. Abstr. (1969) 31, 171. 12 von Hippel, P. Η., Waugh, D. F., J. Am. Chem. Soc. (1955) 77, 4311. 13 Muller, L. L . , Dairy Sci. Abstr. (1971) 33, 659. 14 Borst, J. R., Food Technol. in Australia (1971) 23, 544. 15 Ogden, L. V., Walstra, P., Morris, Η. Α., J. Dairy Sci. (1976) 59, 1727. 16 Timasheff, S. Ν., "Symposium on Foods: Proteins and Their Reac tions," 179-208, AVI Pub. Co., Inc., Westport, Conn., 1964. 17 Nielsen, Μ. Α., Coulter, S. T., Morr, C. V., Rosenau, J . R., J. Dairy Sci. (1973) 56, 76. 18 Morr, C. V., Food Technol. (1976) 30, 18. 19 Morr, C. V., Swenson, P. E . , Richter, R. L . , J. Food Sci. (1973) 38, 324. 20 Panzer, C. C., Schoppet, E. F., Sinnamon, H. I., Aceto, N. C., J. Food Sci. (1976) 41, 1293. 21 Seibles, T. S., Cereal Foods World (1975) 20, 487. 22 Richert, S. H., Morr, C. V., Cooney, C. M., J. Food Sci. (1974) 39, 42. RECEIVED

October 23, 1978.

Functionality and Protein Structure - American Chemical Society

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